The broad, long-term objective of our research is to determine the mechanism of action of parathyroid hormone (PTH) in bone, with the aim of attenuating its short-term catabolic effects on resorption and enhancing its long-term anabolic effects on osteoprogenitor cell recruitment and matrix synthesis. Our goal is to develop new strategies to enhance and maintain peak bone mass or to reduce age-associated losses in skeletal mass, density, and biomechanical strength. The net result of these efforts could be the development of improved treatments for osteoporosis and other metabolic bone diseases that will decrease fracture rates, reduce medical care costs, and improve the quality of life for aging Americans. PTH stimulates osteoblastic and bone-lining cells to retract, exposing the underlying mineral to the resorptive actions of the osteoclast. Thus, osteoblasts play an important role in regulating PTH-dependent resorption by determining the degree of osteoclastic access to the bone surface. PTH stimulates osteoblastic calcium-activated neutral proteases (CANPs) or calpains I and II (calcium-activated papain-like proteases), and 50% of the osteoblastic retraction induced by PTH can be ablated with E64d (a membrane-permeable cysteine protease inhibitor active against CANPs). We hypothesize that the calpain system (calpainI, calpain II, calpastatin [the endogenous inhibitor], and other activators or inhibitors) is present in the osteoblast, where it plays a role in mediating some of the effects of PTH by catalyzing regulatory lysis of cytoskeletal and regulatory proteins and enzymes. Our specific aims are to: (1) Verify the identity of the cysteine protease(s) that mediate PTH-induced osteoblastic retraction by 125I-substrate affinity labeling of PTH-activated proteases in intact MC3T3-E1 cells. The radiolabeled target proteases will be separated by SDS-PAGE and visualized by autoradiography. Their molecular weights will be compared to those of CANP I & II on parallel sets of Western blots. (2) Establish the relative roles of each of the CANPs in mediating PTH- induced osteoblastic retraction by pretreating intact cells with antisense oligonucleotides specific for CANP I, CANP II or CANP I & II. Inhibition of target protein expression will be verified by Western blotting. PTH-induced retraction will be measured by image analysis, and the degree of inhibition of retraction will be examined as a function of CANP protein expression. (3-4) Separate the proteases and their regulatory molecules in MC3T3-E1 cell homogenates by ion exchange FPLC. Determine the absolute and relative amounts of CANP I, CANP II, and calpastatin protein and activity by Western blotting and enzymatic assay. Characterize any CANP activator proteins. This approach is necessary because many different monomeric and dimeric CANPs and tissue-specific activators have been described, and antibodies are not available to probe for all of them. In addition, there is not a direct relationship between the amounts of CAMP proteins present and their enzymatic activities, which are determined by low- and high-molecular weight modulators, posttranslational modification, and subcellular distribution.